Positive data from the CAPRISA 004 trial of tenofovir gel have stimulated tremendous interest in development of HIV-1 entry inhibitors as topical microbicides or even systematically delivered prophylactic drugs to prevent the sexual transmission of HIV-1. While considerable effort has gone into discovering and developing small-molecule agents that inhibit HIV-1 envelope glycoprotein (Env)-mediated virus-cell fusion by targeting the gp41 subunit, none of these compounds has successfully advanced in clinical development of topical microbicides. The long-term objective of this research plan is to develop a safe and highly effective low-molecular-weight gp41 fusion inhibitor for use as an active ingredient in anti-HIV-1 microbicides. In preliminary work, we have identified the novel target of the HIV-1 fusion inhibitor phenyl sulfonamide derivative (PSD) that was first identified in an Env-mediated cell-cell fusion inhibitor screen. This compound binds to a conserved hydrophobic pocket within a subdomain of gp41. We demonstrate that new PSD analogs potently inhibit in vitro infection of human T cells by primary HIV-1 isolates with different geographic origins and co-receptor requirements. We have defined a provisional structure-activity relationship (SAR) for PSDs. The structural data and SAR suggest PSD modifications that could improve potency and pharmacological properties. We propose to leverage the structural data, SAR and synthetic procedures developed in preliminary work to design, synthesize and evaluate novel PSD analogs that have enhanced efficacy against a broad-spectrum of sexually transmitted drug-sensitive and drug-resistant HIV-1 strains. To achieve our goals we have developed the following specific aims: 1. To utilize the structural principles of gp41 fusion inhibitor-target interactions to optimize the antiviral potency and microbicidal properties of PSDs. 2. To evaluate the potency, breadth and synergistic interactions, the molecular basis for antiviral activity and resistance, the physicochemical properties and the in vitro and in vivo toxicity of novel PSDs. 3. To evaluate the effectiveness of novel PSDs in mucosal protection against HIV-1 infection in vitro and in vivo. These aims are motivated by the opportunity to validate the novel gp41 hydrophobic pocket as the target of inhibitor binding, based on our identification of specific inhibitor-target site interactions that underlie structure-based antagonism of HIV-1 gp41 function in cell entry and that can be strengthened to improve potency and broad-spectrum activity. Our ability to piece together a quantitative picture of these inhibitor-target interactions sets the stge for rational development of new small-molecule gp41 fusion inhibitors for prophylactic and/or therapeutic intervention of HIV-1 infection.